Scalable bio-element analysis

ABSTRACT

A method is provided for detecting one or more analytes in a sample. The method relies, in part, on the ability of functionalized particles added to the sample to partially or completely inhibit the transmission of electromagnetic radiation into and out of the sample through a detection surface in a reaction vessel containing the sample. In a microarray format, the invention can be used to screen millions, billions or more biological elements, such as an organism, cell, protein, nucleic acid, lipid, saccharide, metabolite, or small molecules. Methods, apparatuses and kits are described.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.13/791,967 filed Mar. 9, 2013, which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/667,930 filed Jul. 3, 2012,which is incorporated by reference herein in its entirety.

BACKGROUND Technical Field

The invention relates to the detection of one or more analytes in asample or a series of samples. More particularly, the invention relatesto a method for determining one or more biological elements, e.g.,biological cells, in a population of biological elements.

Related Art

Using conventional technologies, biological libraries may be screenedfor components containing, for example, cells, antibodies, proteins,peptides and, nucleic acids. These technologies include phage display,ribosome display, yeast, bacterial display, in vitrocompartmentalization, microengraving and spatial addressing. Suchsystems have a number of disadvantages, including the need to enrich fordesired clones via repeat selection steps (including, for example“panning”) that inherently result in the loss of potential bindingcandidates. It is also difficult to establish the precise origin of apositive signal using conventional technologies since they obtain mixedsignals from heterogeneous populations that cannot be deconvoluted.Generally these techniques involve selection processes utilizingbacteriophages, ribosomes and specific cells, most of which areperformed in vitro.

Improvements in library screening have introduced the concept of spatialaddressing in order to maintain identity of the screened componentsduring the selection process. Such addressing can be based upontechniques including robotics, enzyme-linked immunosorbent assays, orcell-based assays. While spatial addressing can, for example, identifyspecific cellular clones to generate master stocks, these approaches donot facilitate high throughput screening techniques to selectivelyisolate and purify the identified clones for rapid application todisease diagnostics and therapeutics. Another disadvantage of thepresent screening assays is that they are usually limited to a cellnumber between approximately 50 and 100 thousand.

For performing cell-based screening, one of the particular challenges isthe isolation of small populations of cells in a manner that allows forsubsequent screening procedures. Traditional devices and methods ofisolating cells do not adequately provide for the isolation of smallpopulations of cells without performing steps that potentially modifycellular function or activity. Isolation of cells is not only importantin screening, but also in processes that involve the monitoring,measuring, and/or use of the output of cellular activity or function(e.g., antibody production) for small populations of cells.

Accordingly, the need to isolate small numbers of specific cells frombackground populations is ubiquitous, with applications in pathology,clinical diagnosis, cloning, and cell biology research. Currentscreening methods have numerous technical challenges, including: thesize of protein displayed has to be small, the multiplicity of infection(MOI) needs to be high to avoid loss of diversity, the dependency on theactivity of the phage, multiple panning rounds are needed (taking up to1 week or more), high non-specific binding due to phage, antibodies maynot function well in soluble form (truncated clones are oftenexpressed), and/or avidity effects can hinder selection of high affinityclones.

Accordingly, the inventors have identified a need in the art for a moreefficient process of identifying, isolating and characterizingcomponents of biological populations.

SUMMARY

In one aspect, the invention is directed to a method for detecting ananalyte in a sample. The method relies, in part, on the ability ofmicroparticles in a sample to partially or completely inhibit thetransmission of electromagnetic radiation into and out of the samplethrough a detection surface in a vessel containing the sample. In oneembodiment, the method includes adding to a reaction vessel containing asample solution, first particles, and a first label that emitselectromagnetic radiation, wherein the first label is bound to the firstparticles or the first label becomes bound to the first particles as aresult of the presence or absence of the analyte in the sample. Thefirst particles are accumulated at a first detection surface to inhibitthe transmission of electromagnetic radiation into and out of the samplethrough the surface. The presence or amount of electromagnetic radiationemitted from the first detection surface is detected. The firstparticles may accumulate at the detection surface as a result of a forceapplied to the sample, wherein the force is selected from gravitational,magnetic, electrical, centrifugal, convective and acoustic forces. Inone aspect, the label is bound to the particle and is released as aresult of the presence or absence of the analyte in the sample.

In a further embodiment, the method of the invention includes adding tothe vessel a second label that emits electromagnetic radiation andsecond particles that are different from the first particles based uponat least shape, size, density, magnetic permittivity, charge, andoptical coating, wherein the second label is bound to the secondparticles or the second label becomes bound to the second particles as aresult of the presence or absence of a second analyte in the sample. Thesecond particles are accumulated at a second detection surface toinhibit the transmission of electromagnetic radiation into and out ofthe sample through the second detection surface. The presence or amountof electromagnetic radiation emitted at the second detection surface isdetected. In various aspects of this embodiment, the first particlesaccumulate at the first detection surface as a result of a first forceand the second particles accumulate at the second detection surface as aresult of second force, wherein the first force and the second force areindependently selected from gravitational, magnetic, electrical,centrifugal, convective and acoustic forces, and wherein the first forceand the second force are applied to the sample either simultaneously orsequentially. Alternatively, the second particles may be accumulated atthe first detection surface to inhibit the transmission ofelectromagnetic radiation into and out of the sample through the firstdetection surface, wherein the first particles accumulate at the firstdetection surface as a result of a first force and the second particlesaccumulate at the first detection surface as a result of second force,wherein the first force and the second force are independently selectedfrom gravitational, magnetic, electrical, centrifugal, convective andacoustic forces, and wherein the first force and the second force areapplied to the sample sequentially. The presence or amount ofelectromagnetic radiation emitted from the first particles and thesecond particles can be detected at the first detection surface.

Still further, the invention is directed to a method for detecting atarget biological element from a heterogeneous population of biologicalelements The method includes distributing the heterogeneous populationof biological elements into an array of receptacles; adding to the arrayparticles and a first label that emits electromagnetic radiation uponactivation, wherein the first label is bound to the first particles orthe first label becomes bound to the first particles as a result of thepresence or absence of the analyte in the sample; applying a force tothe array to accumulate the particles at a surface of the sample in eachof the receptacles; and identifying the presence or amounts ofelectromagnetic radiation emitted by the receptacles, therebyidentifying receptacles containing the target biological element. Thearray of receptacles may be a microcavity array having a plurality oflongitudinally fused capillaries that have a diameter of about 1 toabout 500 micrometers. In addition, the array may have between about 300and 64,000,000 of the capillaries per square centimeter of the array.The sample may be added to the array at a concentration that is intendedto introduce no more than a single biological element in eachreceptacle.

In various embodiments of the invention, the vessels or receptables aremicrocavities having detection surfaces that are the meniscuses of thesample solution in the microcavities. The sample may be mixed byapplying a magnetic field to move the particles within the samplesolution, wherein the particles are magnetic.

In addition, the biological element may be an organism, cell, protein,nucleic acid, lipids, saccharides, metabolite, or small molecule. Forinstance, the cell produces a recombinant protein such as an enzyme orantibody.

Still further, the invention is directed to a method of extracting asolution including a biological element from a single microcavityassociated with an electromagnetic radiation absorbent material in amicrocavity array. The method includes focusing electromagneticradiation at the microcavity to generate an expansion of the material orthe sample or an evaporation of the sample that expels at least part ofthe sample from the microcavity. In various embodiments, materialincludes particles in the microcavity or the material is a high thermalexpansion material at least partially coating or covering themicrocavity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram of a representative embodiment of themethods disclosed herein.

FIG. 2 illustrates an example of one embodiment of a high density arrayas used according to the disclosure.

FIG. 3 illustrates one embodiment of adding or loading a sample solutioncomprising the analyte and other biological elements to an arraycontainer.

FIG. 4 illustrates one embodiment of a method for scanning andidentifying the pores that produce signal. These pores will appear ashigh intensity spots, due to the accumulation of labelled particles atthe detection surface of the pore. In one assay design as embodiedherein, such as a sandwich assay, pores that have strong signal will beselected for extraction and further preparation. In another assay designas embodied herein, such as an enzyme activity assay, the pores thathave strong signal will not be selected. Instead the pores that show theleast amount of signal, or no signal, will be selected.

FIG. 5 illustrates the ability of the particles to inhibit the signalfrom labels in solution that are not bound to the particles.

FIG. 6 illustrates that a protein binding and detection assay can beperformed directly in the pores as disclosed herein. In this embodiment,all of the pores of the array comprise fluorescently labelled (Atto 590)streptavidin and magnetic beads coated with biotin (Part A: Positivecontrol) and Oligo(dT)₂₅ (Part B: Neg. Control).

FIG. 7 demonstrates the specificity of the disclosed methods at thesingle cell level. In this embodiment, all of the pores of the arraycomprise E. coli cells expressing recombinant protein GFP. Each wellalso contains magnetic beads coated with a rabbit anti-GFP antibody(Pos. Ctrl) and magnetic beads coated with Oligo(dT)₂₅ (Neg. Ctrl).

FIG. 8 demonstrates that the methods disclosed herein do not requirecell lysis for analyte detection. In these embodiments, the arrays wereloaded with E. coli cells expressing recombinant protein GFP. Each wellalso contains fluorescent labels (Atto 590), which is also linked to arabbit anti-GFP antibody. The two positive control wells containmagnetic beads linked to anti-GFP antibody, but the cells of one arraywere lysed using sonication prior to detection. The negative controlarrays were lysed using sonication, and contain Oligo-dT beads, whichuniversally inhibit the signal from all pores in the array

FIG. 9 is a schematic diagram of one method of single pore extraction.

FIG. 10 demonstrates that reagents, particles, or other molecules can beadded or subtracted from the array containing cells without disturbingthe cells. Propidium iodide (PI) was loaded into the micropore arraywithout disturbing the preloaded HEK 293 cells. Part A: Before PIloading with minimal background signal. Part B: After loading PI thathas intercalated into the nucleuses of the non-viable HEK 293 cells.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The invention is directed to methods, apparatus, and kits for detectingan analyte in a sample. In various embodiments, the invention isdirected to the screening of large populations of biological elementsfor the presence or absence of subpopulation of biological elements or asingle element. The invention can be used to discover, characterize andselect specific interactions from a heterogeneous population of millionsor billions of biological elements.

The invention uses biological assay techniques wherein functionalizedparticles accumulate at a surface of a sample vessel and physicallyinhibit, either partially or completely, the transmission ofelectromagnetic radiation into and/or out of a sample liquid. In aspecific embodiment, a high-density microcavity, e.g., micropore, arrayis screened by detecting an electromagnetic signal emitted from a labelin each cavity. High sensitivity can be accomplished with an array thatis arranged such that each cavity contains a single or a few biologicalelements and microparticles capable of inhibiting electromagnetic (EM)radiation into and/or out of each cavity. Through the use ofmicro-cavity arrays and a homogenous particle-based assay, the method ofthe invention provides for scalable single cell analysis. This methodcan be used to discover very rare (1 in 10⁸) biological elements, e.g.,cells, in large complex mixtures.

The invention provides several advantages over other methods ofscreening populations of biological elements. First, it allows simplescreening of millions, billions or more, of biological interactions inparallel. The invention also allows for the display and independentrecovery of analytes, such as target cells. In addition, it providesconcentration versus affinity information for billions of clones inparallel (e.g., feedback on production efficiency can be provided foreach expressed gene).

In one embodiment, disclosed is a method for selecting a subpopulationantibody producing biological cell clones, e.g., a single or severalcell clones, from a population of thousands, millions, or even billionsof cell clones using a micro-cavity array (for example, a porous glassarray). In one embodiment, the micro-cavity are filled with a solution(e.g., a culture media) containing biological cell clones harboringantibody (or any protein of interest) producing genes. The cells growand express antibodies into the media, which can react and bind with abinding partner that is immobilized on a particle within the media. Anantigen-antibody complex can be detected by adding fluorescent reagents(e.g., fluorescently-labeled anti-analyte antibody) to the media.

In one embodiment, the method includes recovering the biological cellsfrom the micropore array. In one embodiment, the biological cellscomprise cells producing a fluorescent protein. In one embodiment, thebiological cells comprise cells producing a fluorescent protein fused toa non-fluorescent protein.

The present invention may be used to isolate any types of biologicalcells, including, but not limited to, cell lines that express or produceproteins, carbohydrates, enzymes, peptides, hormones, receptors; othercell lines that produce antibodies; genetically engineered cells; andactivated cells. Moreover, the present invention may be used to screenfor a variety of biological activities including, but not limited to,the expression of surface receptor proteins, enzyme production, andpeptide production. Furthermore, the present invention may be used toscreen a variety of test agents to determine the effect of the testagents on the desired biological activity. Other types of cells desiredto be isolated and screened, other types of biological activity desiredto be detected, and specific test agents to be screened will be readilyappreciated by one of skill in the art.

Definitions

Unless otherwise defined, the technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Expansion and clarification of some terms are provided herein.All publications, patent applications, patents and other referencesmentioned herein, if not otherwise indicated, are explicitlyincorporated by reference.

As used herein, the singular forms “a,” “an”, and “the” include pluralreferents unless the context clearly dictates otherwise.

The terms “binding partner”, “ligand” or “receptor” as used herein, maybe any of a large number of different molecules, or aggregates, and theterms are used interchangeably. In various embodiments, the bindingpartner may be associated with or bind an analyte being detected.Proteins, polypeptides, peptides, nucleic acids (nucleotides,oligonucleotides and polynucleotides), antibodies, saccharides,polysaccharides, lipids, receptors, test compounds (particularly thoseproduced by combinatorial chemistry), may each be a binding partner.

The term “biological cell”, refers to any cell from an organism,including, but not limited to, viral, insect, microbial, fungal (forexample, yeast) or animal, (for example, mammalian) cells.

The term “biological element” as used herein, refers to any bioreactivemolecule. Non-limiting examples of these molecules include proteins,nucleid acids, peptides, antibodies, antibody fragments, enzymes,hormones, biological cells, and small molecules.

An “analyte” generally refers to an element of interest in a sample, forexample a biological element of interest in a biological sample.

The term “bind” or “attach” as used herein, includes any physicalattachment or close association, which may be permanent or temporary.These attachments or close associations may be interactions.Non-limiting examples of these associations are hydrogen bonding,hydrophobic forces, van der Waals forces, covalent bonding, and/or ionicbonding. These interactions can facilitate physical attachment between amolecule of interest and the analyte being measured. The “binding”interaction may be brief as in the situation where binding causes achemical reaction to occur, such as for example when the bindingcomponent is an enzyme and the analyte is a substrate for the enzyme.

Specific binding reactions resulting from contact between the bindingagent and the analyte are also within this definition. Such reactionsare the result of interaction of, for example, an antibody and, forexample a protein or peptide, such that the interaction is dependentupon the presence of a particular structure (e.g., an antigenicdeterminant or epitope) on a protein. In other words an antibody isrecognizing and binding to a specific protein structure rather than toproteins in general. For example, if an antibody is specific for epitope“A”, the presence of a protein containing epitope A (or free, unlabelledA) in a reaction containing labeled “A” and the antibody will reduce theamount of labeled A bound to the antibody. Specific binding interactionscan occur between other molecules as well, including, for example,protein-protein interactions, protein-small molecule interactions,antibody-small molecule interactions, and protein-carbohydrateinteractions. Each of these interactions may occur at the surface of acell.

The terms “arrays” and “microarrays” are used interchangeably differingonly in general size. In various embodiments, the arrays typicallycontain a multitude (typically 100 to over 1,000,000) of distinctreaction spaces, for pores, wells, cavities, containers or receptacles,wherein each vessel can be at a known location and contain a single ornumerous components of interest.

The term “sample” as used herein is used in its broadest sense andincludes environmental and biological samples. Environmental samplesinclude material from the environment such as soil and water. Biologicalsamples may be animal, including, human, fluid (e.g., blood, plasma andserum), solid (e.g., stool), tissue, liquid foods (e.g., milk), andsolid foods (e.g., vegetables). For example, a pulmonary sample may becollected by bronchoalveolar lavage (BAL), which comprises fluid andcells derived from lung tissues. A biological sample may comprise acell, tissue extract, body fluid, chromosomes or extrachromosomalelements isolated from a cell, genomic DNA, RNA, cDNA and the like.

DESCRIPTION OF VARIOUS EMBODIMENTS

Disclosed is a method for detecting an analyte in a sample. The methodincludes adding to a vessel having a defined detection surface a samplesolution containing the analyte, particles, and a label that emitselectromagnetic radiation. The particles can accumulate at the detectionsurface as a result of a force applied to the vessel and inhibitemission of a signal from the label in the sample other than the labelthat may be attached to the particles accumulated at the detectionsurface. The presence or absence of the analyte in the sample controlswhether the particles can accumulate at a detection surface of thevessel and/or whether a label that may be bound to the particles at thesurface can emit electromagnetic radiation. The invention can be usedwith a variety of assay formats known in the art.

In one aspect, the label is bound to the particles or the label becomesbound to the particles as a result of the presence or absence of theanalyte in the sample. In accordance with this embodiment of thedisclosure, the particles may be functionalized with a binding partnerthat binds the analyte, which can be analyzed with assay formats wellknown to those of skill in the art, e.g., sandwich and competitiveimmunoassay methods. In a sandwich method, the particle isfunctionalized with a binding partner for the analyte and is mixed witha label that includes a moiety capable of emitting a signal (e.g.,fluorescent moiety) and a binding partner for the analyte. The labelbecomes bound to the particle as a result of the binding of the analyteto the particle and the label. The presence of the label on the particleindicates the presence of the analyte in the sample. In a competitivedetection format, particles having a binding partner for the analyte anda label that includes second binding partner that is an analogue of theanalyte are mixed with the sample. The analogue binds in competitionwith the analyte in the sample to the binding partner for the analyte onthe particle. The absence of signal from the label indicates thepresence of the analyte in the sample.

In another assay format, the analyte is an enzyme, and the particle isfunctionalized directly with a substrate for the enzyme (e.g., bycovalent binding) that acts as a label, or the enzyme is bound to theparticle as a result of specific or non-specific binding, or otherinteraction. The presence or absence of signal from the enzyme/label isindicative of the activity of the analyte/enzyme to convert the labelfrom signal producing to non-signal producing, or vice versa.

Another assay format uses the analyte's ability to release a label boundto a particle. For example, the analyte may be an enzyme that cleaves alinker between the label and the particle. Alternatively, the analyte,when present in the sample may prevent cleavage of the linker andrelease of the label from the particle.

After the binding or enzyme reaction is allowed to proceed, theparticles are accumulated at a detection surface to inhibit, eitherpartially or completely, the transmission of electromagnetic radiationinto and out of the sample through the surface. The presence or amountof electromagnetic radiation at the detection surface is thendetermined. Accordingly, when the particles are accumulated at thesurface, electromagnetic radiation from the label attached to theparticles can be detected at the surface. The particles, however, act asa shutter at the surface to inhibit electromagnetic radiation from labelthat is not bound the particles. Accordingly, the background signal ofunbound label in the sample is eliminated. Similarly, when the particlesdo not have any bound label, the particles will not emit anyelectromagnetic radiation at the detection surface and act as a shutterto inhibit signal from unbound label in the sample solution from beingdetected at the surface.

In another embodiment, a reaction vessel includes a binding partner onthe wall of the vessel. In a binding reaction as a result of thepresence or absence of the analyte, the particles are captured, or not,on the surface of the reaction vessel. The particles do not then inhibittransmission of electromagnetic radiation from a label in the sample outof the reaction vessel through a detection surface. In this embodiment,the label is not required to participate in the binding reaction and canremain unbound in solution. The binding reaction controls the ability ofthe particles to accumulate at the detection surface when a forceapplied to the reaction vessel. In this embodiment, particles may becomebound to the surface of the vessel in the presence of the analyte whenthe particles and the wall of the vessel are coated with bindingpartners for the analyte, wherein the analyte becomes sandwiched by thebinding partners. In this embodiment, a signal from the label in thereaction vessel indicates the presence of the analyte because theparticles become bound to wall of the reaction vessel and are unable toinhibit electromagnetic radiation from exiting the vessel. In acompetitive assay format, a particle coated with an analyte analoguewould bind to a vessel wall coated with a binding partner for theanalyte in the absence of the analyte. Therefore the absence of a signalfrom the vessel would indicate the presence of the analyte.

When the label is activatable by an external source, e.g., a fluorescentlabel excitable by electromagnetic radiation, the particles accumulatedat the surface will prevent the electromagnetic radiation from excitingany label other than label bound to particles at the surface. Theparticles at the detection surface act as a shutter to preventelectromagnetic radiation from entering the sample solution and excitingunbound label in solution. Accordingly, only label that is attached tothe particles at the surface are excited and background signal fromunbound label is avoided. When the label is activatable by a sourceother than electromagnetic radiation (e.g., heat, electricity, chemicalreaction, enzymatic reaction), the electromagnetic radiation fromactivated label in the sample is prevented from exiting the vesselthrough the detection surface because of particles accumulated at thedetection surface.

The size of the reaction vessel is limited only by the ability of theparticles to accumulate at a reaction surface of the vessel and inhibit,either partially or completely, the transmission of electromagneticradiation in to and out of the vessel. Smaller formats are preferred assmaller sample sizes are necessary for screening multitudes ofbiological elements in an efficient manner.

Reaction vessels suitable for use with the invention have or can beconstructed to have a detection surface where the functionalizedparticles can be accumulated. The surface is not limited to particularmaterial as long as it is transmissive to electromagnetic radiation.Alternatively, the surface is not bound by any material and may be opento the environment such that the detection surface is the surface of thesample solution, for instance the meniscus of the sample solution in areaction well, microwell, micropore, capillary or microcavity.Transmission of electromagnetic radiation through the surface should beunimpeded except when the particles are accumulated at the surface. On amacroscale, the surface may be a window in a cuvette or reaction well.Regardless of vessel size, the area surrounding the surface shouldprevent the transmission of electromagnetic radiation in to and out ofthe sample such that electromagnetic radiation can enter the sample onlythrough the surface. A reaction vessel may have more than one surface,such as, for example, multiple windows or a meniscus at each open end ofa capillary or microtube.

Accumulation of particles at the detection surface can be accomplishedby applying a force to the sample that draws the particles to thesurface. The force should be sufficient such that the accumulation ofparticles prevents at least 50% of the electromagnetic radiation frompassing through the accumulated particles. In particular embodiments,electromagnetic radiation is inhibited from entering or exiting thesample through the detection surface when at least 30%, 40%, 50%, 60%,70%, 80%, 90%, 95% or 100% of the radiation is prevented from enteringor exiting the sample through the surface. Exemplary types of forcesthat can be used to accumulate particles at the detection surfaceinclude gravitational, magnetic, electrical, centrifugal and acousticalforces.

Gravitational force can be used to allow particles to settle at thebottom of a reaction vessel. When the bottom of the vessel includes adetection surface, the signal from a label associated with the particlescan be detected. Magnetic force applied to the sample containingmagnetic particles can be used to draw the particles to a detectionsurface at the top or other location in the vessel. In specificembodiments, the magnetic force is applied by a magnet, e.g., a cubedneodymium magnet (B666, K&J Magnetics Inc.).

Similarly, charged particles can be used subjected to electrical forcesthat move the particles throughout the sample and towards a detectionsurface. In some embodiments, the force is an electrokinetic force asdescribed, for example, in U.S. patent publication No. 2006/0078998,which is incorporated by reference herein in its entirety. In someembodiments the particles are insulating and inhibit the application ofan electrical force to the sample, thereby preventing the activation ofan electrically stimulated label in the sample.

Likewise, different shaped particles with different resonant frequenciescan be used subjected acoustic wave frequency that displace andaccumulate the resonant particles to the acoustic pressure nodes. Insome embodiments, the force is an acoustic force as described in, forexample, in Laurell, T., et al., Chem. Soc. Rev. 2007, 36:492-506, whichis incorporated by reference herein in its entirety.

FIG. 1 depicts an exemplary antigen-antibody recognition assay, or“sandwich” assay in a micropore array. In this embodiment, the particlesare magnetic microparticles and are associated with and/or bound to theantigen. The analyte as depicted is a target protein that is beingproduced by cells in the sample. A fluorescent label is associated withor bound to an antibody that is specific for and/or binds the targetprotein. Panel (A) is a schematic diagram of the various biologicalcomponents present in one or more pores before any force is applied tothe array. The particles are associated with the fluorescent label dueto antigen binding to the target protein, which is also bound to thelabelled antibody. Panel (A) represents a time point shortly afteraddition of sample to the pore, because some target protein remainsunbound to the antigen and/or antibody. In some embodiments, the arraywould then be incubated and/or stirred to facilitate target proteinbinding. After optional incubation, panel (B) depicts the pore followingthe accumulation of the particles to the detection surface byapplication of a magnetic force. In this embodiment, the detectionsurface is the open surface of the liquid in the pore. In this example,the electromagnetic (EM) signal from the label bound to the magneticparticle is detected from the pore surface.

In additional embodiments, a method is provided for detecting two ormore analytes in a sample. The method includes adding to the reactionvessel a second label that emits electromagnetic radiation and secondparticles that are different from the first particles based upon atleast one of the following properties: shape, size, density, magneticpermittivity, charge, and optical coating. As described above, thesecond label is bound to the second particles or the second labelbecomes bound to the second particles as a result of the presence orabsence of a second analyte in the sample (e.g., an enzyme is bound tothe particles or a label becomes bound as a result of a competitive orsandwich assay). The second particles are accumulated at a seconddetection surface to inhibit the transmission of electromagneticradiation into and out of the sample through the second detectionsurface. The presence or amount of electromagnetic radiation emittedfrom the second particles can be detected at the second detectionsurface.

In various embodiments first particles for detecting a first analyteaccumulate at the first detection surface as a result of a first forceand the second particles accumulate at the second detection surface as aresult of second force, wherein the first force and the second force areindependently selected from the group consisting of gravitational,magnetic, electrical, centrifugal and acoustic force as described above.The first force and the second force are applied to the sample, anddetection can be accomplished either simultaneously or sequentially.

Particles

High sensitivity is achieved by concentrating particles at the detectionsurface when the particles have the ability to shutter the sample, forexample inhibit excitation energy from an in-solution flourochrome andthe transmission of background signal to a detector. Suitable particlesare readily commercially available and a wide variety of particles canbe used according to the methods disclosed herein as long as theparticles can accumulate and inhibit electromagnetic radiation throughthe detection surface. In various embodiments, the particles arepartially or fully opaque. In certain embodiments, the particles absorbelectromagnetic radiation, for example the particles have an efficiencyof absorbance of at least about 10 percent, for example, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 percent.

In various embodiments, the size of the particles ranges from nanoscaleto about one-third the size of the cross section of a reaction vessel.For example, when a microcavity is about 20 microns in diameter, theparticle can be about 0.01 to 7 microns in diameter. The size of theparticles should allow for accumulation and of particles at thedetection surface of the vessel so that the accumulation of theparticles inhibit electromagnetic radiation from entering or exiting thesample solution. For example, when the reaction vessel is a micropore,the sample solution will form a meniscus with the wall of the micropore.When the micropore is open at both ends, a meniscus forms at both thetop and the bottom of the micropore. The particles should be able toaccumulate at the meniscus at the top or bottom of the micropore suchthat electromagnetic radiation is prevented for existing or entering thesample solution in the micropore.

In specific embodiments, the particle diameter ranges from about 0.01microns to about 50 microns, depending on the size of the vessel used.In various embodiments, the particles range in size from about 0.1 to 15microns, about 0.5 to 10 microns, and about 1 to about 5 microns.

In certain embodiments, the particles comprise a metal or carbon.Non-limiting examples of suitable metals include gold, silver, andcopper. Other metallic materials are suitable for use in binding assaysas is well known to those of skill in the art.

In one embodiment, the particles are magnetic such that magnetic forcecan be used to accumulate the particles at the detection surface of eachreaction vessel, e.g., the meniscus of a micro-cavity.

The surface chemistry of the particles may be functionalized to providefor binding to sample components as is well known to those of skill inthe art. For example, the particles are coupled with streptavidin,biotin, oligo(dT), protein A &G, tagged proteins, and/or any otherlinker polypeptides. The very high binding affinity of thestreptavidin-biotin interaction is utilized in a vast number ofapplications. Streptavidin bound particles will bind biotinylatednucleic acids, antibodies or other biotinylated ligands and targets.Biotinylated antigens are a useful example of the products that could bebound to the particles for screening for analytes. In a specificembodiment, the particles are DYANABEAD® particles (Invitrogen,Carlsbad, Calif.) coupled to several different ligands. For example,oligo(dT), protein A &G, tagged proteins (His, FLAG), secondaryantibodies, and/or streptavidin. (Part No. 112-05D, Invitrogen,Carlsbad, Calif.).

In some embodiments, particles having different magnetic permittivitiescan be used to provide independent control of the magnetic forces actingon the particles. In other embodiments, other properties of theparticles can be used to expand the multiplexing capability of theassays done in each cavity. When added to a sample, particles bind tothe desired target (cells, pathogenic microorganisms, nucleic acids,peptide, protein or protein complex etc). This interaction relies on thespecific affinity of the ligand on the surface of the particles.Alternatively, the particles conjugated to substrate for an enzyme canbe added to the sample, where the enzyme/analyte in the sample eitherquenches the ability of the substrate to fluoresce or activates thesubstrate to be fluorescent (e.g., enzyme mediated cleavage of thesubstrate).

Another embodiment uses magnetic particles having different shapes,densities, sizes, charges, magnetic permittivity, or optical coatings.This allows different probes (i.e., binding partners) to be put on thedifferent particles and the particles could be probed separately byadjusting how and when the magnetic field or other force is applied.Sedimentation rates can also be used to separate the particles by size,shape and density and expand the multiplexing capability of the assaysdone in each cavity.

For example, one particle could contain the target antigen which wouldallow determination of the affinity of the antibodies produced in eachcavity to the target. The other particle located in each cavity couldcontain a wide range of antigens on the same particle which allowmeasurement of the specificity of the antibody. The settling times ofparticles having different sizes or densities when no magnetic field canbe exploited. The particles with the faster settling times can bedetected by scanning a detection surface at the bottom of the cavity. Ifthe particles with the slower settling time had a higher magneticpermittivity, they could be attracted to the bottom of the cavity beforethe other particles only when the magnetic field was applied.

Similarly, some of the particles can be magnetic and the others not, andthe magnetic particles can be drawn to the top of the cavities by anapplied magnetic field and there detected, while the nonmagneticparticles settle to the bottom of the cavity and can be detected there.Using these types of methods, both sensitivity and specificity of theantibodies can be measured in the same well.

In certain embodiments, the particles are used to mix the content of thereceptacles. For example, magnetic particles are subjected to andalternating or intermittent magnetic field(s) during an incubation step.The movement and settling of the particles results in the mixing of thecontents of the reaction vessel.

Any suitable binding partner with the requisite specificity for the formof molecule, e.g., a marker, to be detected can be used. If themolecule, e.g., a marker, has several different forms, variousspecificities of binding partners are possible. Suitable bindingpartners are known in the art and include antibodies, aptamers, lectins,and receptors. A useful and versatile type of binding partner is anantibody.

The method for detecting an analyte in a sample disclosed herein allowsfor the simultaneous testing of two or more different antigens per pore.Therefore, in some embodiments, simultaneous positive and negativescreening can occur in the same pore. This screening design improves theselectivity of the initial hits. In certain embodiments, the secondantigen tested can be a control antigen. Use of a control antigen isuseful for normalizing biological element concentration across thevarious pores in the array. A non-limiting example would be using afirst antigen specific for an analyte of interest, and a second antigenthat is non-specific for all proteins, such as the —N or —C terminal.Therefore the results of pores of interest can be quantified bycomparing the signal to total protein concentration.

In some embodiments, the second antigen is associated with secondparticles that are different from the first particles. The particles canvary by least one of the following properties: shape, size, density,magnetic permittivity, charge, and optical coating. The second label cantherefore associate with the second particles as a result of thepresence or absence of a second analyte in the sample, and processedusing motive forces as described below.

In another embodiment, the particles non-specifically bind samplecomponents. For example, particles can be functionalized tonon-specifically bind all protein in a sample, which allows fornormalization of protein content between sample in an array.

Antibodies

The term “antibody,” as used herein, is a broad term and is used in itsordinary sense, including, without limitation, to refer to naturallyoccurring antibodies as well as non-naturally occurring antibodies,including, for example, single chain antibodies, chimeric, bifunctionaland humanized antibodies, as well as antigen-binding fragments thereof.It will be appreciated that the choice of epitope or region of themolecule to which the antibody is raised will determine its specificity,e.g., for various forms of the molecule, if present, or for total (e.g.,all, or substantially all, of the molecule).

Methods for producing antibodies are well-established. One skilled inthe art will recognize that many procedures are available for theproduction of antibodies, for example, as described in Antibodies, ALaboratory Manual, Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988), Cold Spring Harbor, N.Y. One skilled in the art willalso appreciate that binding fragments or Fab fragments that mimicantibodies can be prepared from genetic information by variousprocedures (Antibody Engineering: A Practical Approach (Borrebaeck, C.,ed.), 1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920(1992)). Monoclonal and polyclonal antibodies to molecules, e.g.,proteins, and markers also commercially available (R and D Systems,Minneapolis, Minn.; HyTest Ltd., Turk, Finland; Abcam Inc., Cambridge,Mass., USA, Life Diagnostics, Inc., West Chester, Pa., USA; FitzgeraldIndustries International, Inc., Concord, Mass., USA; BiosPacific,Emeryville, Calif.).

In some embodiments, the antibody is a polyclonal antibody. In otherembodiments, the antibody is a monoclonal antibody.

Capture binding partners and detection binding partner pairs, e.g.,capture and detection antibody pairs, can be used in embodiments of theinvention. Thus, in some embodiments, a heterogeneous assay protocol isused in which, typically, two binding partners, e.g., two antibodies,are used. One binding partner is a capture partner, usually immobilizedon a particle, and the other binding partner is a detection bindingpartner, typically with a detectable label attached. Such antibody pairsare available from several commercial sources, such as BiosPacific,Emeryville, Calif. Antibody pairs can also be designed and prepared bymethods well-known in the art.

In a particular embodiment, the antibody is biotinylated or biotinlabelled. In another embodiment, the antibody is anti-GFP.

In one embodiment, there is a second imaging component that binds allmembers of the analyte of interest non-specifically. Therefore thissignal can be read to normalize the quantity of fluorescence from poreto pore. One example is an antibody that will bind all proteins at the Nor C terminal. For the control component, the microparticles that arebound to the target element can be accumulated at one detection surface,while the microparticles that are bound to the control elementaccumulate at another detection surface. Alternatively, themicroparticles that bind the target and the control can be detected atthe same detection surface by exploiting a difference in the particlesthat allows them to be at the surface at different times. Accordingly,detection of the labels at the detection window can occur sequentially.

Labels

Several strategies that can be used for labeling binding partners toenable their detection or discrimination in a mixture of particles arewell known in the art. The labels may be attached by any known means,including methods that utilize non-specific or specific interactions. Inaddition, labeling can be accomplished directly or through bindingpartners.

Emission, e.g., fluorescence, from the moiety should be sufficient toallow detection using the detectors as described herein. Generally, thecompositions and methods of the invention utilize highly fluorescentmoieties, e.g., a moiety capable of emitting electromagnetic radiationwhen stimulated by an electromagnetic radiation source at the excitationwavelength of the moiety. Several moieties are suitable for thecompositions and methods of the invention.

Labels activatable by energy other than electromagnetic radiation arealso useful in the invention. Such labels can be activated by, forexample, electricity, heat or chemical reaction (e.g., chemiluminescentlabels). Also, a number of enzymatically activated labels are well knownto those in the art.

Typically, the fluorescence of the moiety involves a combination ofquantum efficiency and lack of photobleaching sufficient that the moietyis detectable above background levels in the disclosed detectors, withthe consistency necessary for the desired limit of detection, accuracy,and precision of the assay.

Furthermore, the moiety has properties that are consistent with its usein the assay of choice. In some embodiments, the assay is animmunoassay, where the fluorescent moiety is attached to an antibody;the moiety must have properties such that it does not aggregate withother antibodies or proteins, or experiences no more aggregation than isconsistent with the required accuracy and precision of the assay. Insome embodiments, fluorescent moieties dye molecules that have acombination of 1) high absorption coefficient; 2) high quantum yield; 3)high photostability (low photobleaching); and 4) compatibility withlabeling the molecule of interest (e.g., protein) so that it may beanalyzed using the analyzers and systems of the invention (e.g., doesnot cause precipitation of the protein of interest, or precipitation ofa protein to which the moiety has been attached).

A fluorescent moiety may comprise a single entity (a Quantum Dot orfluorescent molecule) or a plurality of entities (e.g., a plurality offluorescent molecules). It will be appreciated that when “moiety,” asthat term is used herein, refers to a group of fluorescent entities,e.g., a plurality of fluorescent dye molecules, each individual entitymay be attached to the binding partner separately or the entities may beattached together, as long as the entities as a group provide sufficientfluorescence to be detected.

In some embodiments, the fluorescent dye molecules comprise at least onesubstituted indolium ring system in which the substituent on the3-carbon of the indolium ring contains a chemically reactive group or aconjugated substance. Examples include Alexa Fluor molecules.

In some embodiments, the labels comprise a first type and a second typeof label, such as two different ALEXA FLUOR® dyes (Invitrogen), wherethe first type and second type of dye molecules have different emissionspectra.

A non-inclusive list of useful fluorescent entities for use in thefluorescent moieties includes: ALEXA FLUOR® 488, ALEXA FLUOR® 532, ALEXAFLUOR® 647, ALEXA FLUOR® 700, ALEXA FLUOR® 750, Fluorescein,B-phycoerythrin, allophycocyanin, PBXL-3, Atto 590 and Qdot 605.

Labels may be attached to the particles by any method known in the art,including, absorption, covalent binding, biotin/streptavidin or otherbinding pairs. In addition, the label may be attached through a linker.In some embodiments, the label is cleaved by the analyte, therebyreleasing the label from the particle. Alternatively, the analyte mayprevent cleavage of the linker.

Arrays

In one embodiment, the reaction vessels for use with the invention areincluded in an extreme density porous array. For instance, micro-porearrays contemplated herein can be manufactured by bundling millions orbillions of silica capillaries and fusing them together through athermal process. Such a fusing process may comprise the steps includingbut not limited to; i) heating a capillary single draw glass that isdrawn under tension into a single clad fiber; ii) creating a capillarymulti draw single capillary from the single draw glass by bundling,heating, and drawing; iii) creating a capillary multi-multi draw multicapillary from the multi draw single capillary by additional bundling,heating, and drawing; iv) creating a block assembly of drawn glass fromthe multi-multi draw multi capillary by stacking in a pressing block; v)creating a block pressing block from the block assembly by treating withheat and pressure; and vi) creating a block forming block by cutting theblock pressing block at a precise length (e.g., 1 mm).

In one embodiment, the method further comprises slicing the silicacapillaries, thereby forming a very high-density glass micro-pore arrayplate. It will be appreciated that the array of micro-pores for use inthe present invention can be formed by any suitable method. In oneembodiment, the capillaries are cut to approximately 1 millimeter inheight, thereby forming a plurality of micro-pores having an internaldiameter between approximately 1.0 micrometers and 500 micrometers. Inone embodiment, the micro-pores range between approximately 10micrometers and 1 millimeter long. In one embodiment, the micro-poresrange between approximately 10 micrometers and 1 centimeter long. In oneembodiment, the micro-pores range between approximately 10 micrometersand 10 millimeter long. In one embodiment, the micro-pores range betweenapproximately 10 micrometers and 100 millimeter long. In one embodiment,the micro-pores range between approximately 0.5 millimeter and 1 meterlong.

Such processes form a very high-density micro-pore array that may beused in the present invention. In an exemplary array, each micro-porehas a 5 μm diameter and approximately 66% open space (i.e., representingthe lumen of each micropore). In some arrays, the proportion of thearray that is open ranges between about 50% and about 90%, for exampleabout 60 to 75%, such as a micro-pore array provided by Hamamatsu thathaving an open area of about 67%. In one particular example, a 10×10 cmarray having 5 μm diameter micropores and approximately 66% open spacehas about 330 million micro-pores. See, e.g., FIG. 2.

In various embodiments, the internal diameter of micro-pores rangesbetween approximately 1.0 micrometers and 500 micrometers. In somearrays, each of said micro-pores can have an internal diameter in therange between approximately 1.0 micrometers and 300 micrometers;optionally between approximately 1.0 micrometers and 100 micrometers;further optionally between approximately 1.0 micrometers and 75micrometers; still further optionally between approximately 1.0micrometers and 50 micrometers, still further optionally, betweenapproximately 5.0 micrometers and 50 micrometers.

In some arrays, the open area of the array comprises up to 90% of theopen area (OA), so that, when the pore size varies between 10 μm and 500μm, the number of micro-pores per cm of the array varies between 458 and1,146,500, as is represented in the table below. In some arrays, theopen area of the array comprises about 67% of the open area, so that,when the pore size varies between 10 μm and 500 μm, the number ofmicro-pores per square cm of the array varies between 341 and 853,503,as is represented in the table below. It will be appreciated that, witha pore size of 1 μm and up to 90% open area, each square cm of the arraywill accommodate up to approximately 11,466,000 micro-pores.

Pore diameter (um) No of pore (90% OA) No of pores (67% OA) 500 458 341300 1275 948 100 1150 8535 75 20,380 15,172 50 45.860 34,140 101,146,500 853,503 1 11,465,967 8,535,031

In one particular embodiment, a micropore array can be manufactured bybonding billions of silica capillaries and then fusing them togetherthrough a thermal process. After that slices (0.5 mm or more) are cutout to form a very high aspect ratio glass micro perforated array plate.See, International Application PCT/EP2011/062015 (WO2012/007537), filed13 Jul. 2011 date, which is incorporated by reference herein in itsentirety. Arrays are also commercially available, such as from HamamatsuPhotonics K. K., (Part No. J5022-19) and other manufacturers. In someembodiments, the micropores of the array are closed at one end with asolid substrate attached to the array.

Thus, in addition to being faster and easier to use, the ability todetect produced proteins allows this micro-pore array to provide higherresolution than current methods that rely upon the target molecule beingexpressed on the surface of a display vector (i.e. phage display,ribosome display, mammalian cell display, bacterial cell display oryeast display). Additional benefits of this array as compared to phagedisplay methods include the ability to simultaneously test two (or more)target molecules per pore (i.e. positive and negative screening) and notbeing limited by the size of the protein being examined sincephage-displayed proteins have to be small.

In another embodiment, the present invention contemplates a method forloading array comprising contacting a solution comprising a plurality ofcells with the array to form a loaded array. In one embodiment, loadinga mixture of antibody secreting cells, e.g., E. coli, evenly into allthe micro-pores comprises placing a 500 μL droplet on the upper side ofthe array and spreading it over all the micro-pores. The heterogeneouspopulation of cells can be loaded onto the micro-pore array. In oneembodiment, an initial concentration of approximately 10⁹ cells in the500 μL, droplet results in approximately 3 cells (or sub-population) permicro-pore. In one embodiment, each micro-pore has an approximate volumeof between 20-80 pL (depending on the thickness of the glass capillaryplate of between 250 μm to 1 mm). Once the micro-pores are loaded andincubated overnight, each micro-pore should then contain approximately2,000-3,000 cells per micro-pore. In one embodiment, the cells may becultivated for up to forty-eight hours without loss of viability inorder to maximize the proliferation yield. Although it is not necessaryto understand the mechanism of an invention, it is believed that“spreading” the droplet over all the micro-pores provides for optimaldistribution of cells in the various micro-pores. Theoretically, addinga drop to the micro-pore array should fill all pores evenly. However, anempirical evaluation demonstrated that surface tension actually preventsthe drop from entering the central micro-pores. If the drop is spreadevenly over the micro-pore array surface the surface tension is removed.Consequently, if the drop is placed straight down on the micro-porearray, only the pores at the edge of the drop fill due to reducedsurface tension (also evaporation recedes the drop so that the liquid isno longer held in suspension). This causes a halo ring effect followingdetection of the appropriate analyte. See, WO2012/007537.

In one embodiment, the solution comprises approximately three (3)microliters. In one embodiment, the plurality of cells may be selectedfrom the group comprising animal cells, plant cells, and/or microbialcells. In one embodiment, the plurality of cells comprise E. coli cells.In one embodiment, the E. coli cells secrete at least one recombinantcompound of interest. In one embodiment, the recombinant compound ofinterest has an affinity for the binding partner.

Although it is not necessary to understand the mechanism of aninvention, it is believed that, if there are approximately 10⁹ cells inan approximate 500 μL·solution then, on average, there should beapproximately three (3) cells per micro-pore for an array havingapproximately 3-4×10⁶ micro-pores. It should be noted that the exactnumber will depend on the number of pores in the array. For example, ifan array has approximately 3-4×10⁶ micro-pores, it therefore, would haveapproximately 500-100 cells/pore. In one embodiment, each micro-porecomprises a volume of ranging between approximately 20-80 pico liters.

In certain embodiment, the sidewalls of the cavities of the arrays arenot transmissive to electromagnetic radiation, or the cavities arecoated with a material that prevents the transmission of electromagneticradiation between cavities of the arrays. Suitable coating should notinterfere with the binding reaction within the cavities or theapplication of forces to the cavities. Exemplary coatings includesputtered nanometre layers of gold, silver and platinum.

In some embodiments, the cavities of the array have a hydrophilicsurface which can spontaneously uptake the solution into the pore.

Sample Dilution, Preparation, Incubation

The sample containing the population and/or library of biologicalsamples may require preparation steps prior to distribution to thearray. In some embodiments, these preparation steps include anincubation time. The incubation time will depend on the design of thescreen. Example times include 5 minutes, 1 hour, and/or 3 hours. Exampleranges are 1 second to 72 hours.

In certain embodiments, the heterogeneous population of biologicalelements is expanded in media prior to adding and/or loading onto thearray. For certain applications, the media and element mixture is loadedinto the array while in the exponential growth phase.

In other embodiments, the sample containing the heterogeneous populationand/or library of biological samples may require preparation steps afteraddition to the array. In other embodiments, each element within eachpore is expanded (cells grown, phages multiplied, proteins expressed andreleased, etc.) during an incubation period. This incubation periodallows the biological elements to produce bioreactive molecules.

For some embodiments, each pore has a volume of media that will allowcertain biological elements to replicate. In specific embodiments, thevolume of media is about 20 picoliter, which provides sufficient mediato allow most single cells within a pore to replicate multiple times.The array can optionally be incubated at any temperature, humidity, andtime for the biological elements to expand and produce the targetproteins. Incubation conditions can be determined based on experimentaldesign as is routine in the art.

Concentration, Conditions, Loading

In a specific embodiment, the array is designed such that some or allpores contain a single biological element to screen for the analyte. Theconcentration of the heterogeneous mixture of biological elements istherefore calculated according to the design of the array and desiredanalytes to identify. In embodiments where protein-producing cells arebeing screened, the method is advantageous because it eliminates theclonal competition and thus can screen a much larger diversity.

In one embodiment, the method of the present invention contemplates theconcentration of the suspension of heterogeneous population of cells andthe dimensions of the array are arranged such that 1-1000 biologicalelements, optionally, 1-500 biological elements, further optionally,1-100 biological elements, still further optionally 1-10 biologicalelements, still further optionally, 1-5 biological elements, aredistributed into at least one of said micropores of the array.

The volume of the drop will depend on several variables, including forexample the desired application, the concentration of the heterogeneousmixture, and/or the desired dilution of biological elements. In onespecific embodiment, the desired volume on the array surface is about 1microliter per square millimeter. The concentration conditions aredetermined such that the biological elements are distributed in anydesired pattern or dilution. In a specific embodiment, the concentrationconditions are set such that in most pores of the array only singleelements are present. This allows for the most precise screening ofsingle elements.

These concentration conditions can be readily calculated. By way ofexample, in a cell screen, if the ratio of protein-producing cells topores is about 1 to 3, an array with 10⁹ pores could be loaded with3×10⁸ different protein-producing cells in a 6 mL volume (6 mL=20picoliter/pore×3×10⁸ pores), the vast majority of the pores will containat most a single clone. In certain other embodiments, single biologicalelements are not desired in each pore. For these embodiments, theconcentration of the heterogeneous population is set so that more thanone biological element is found in each pore. For example, FIG. 3 showsthe addition of a mixture of protein-producing cells (bacterial cells:E. coli) being loaded into the pores of an array by placing a 1.0 mLdrop on one side of the array and spreading it over all of the pores.

The screening may also be used to enrich a population of biologicalelements, such as biological cells. For instance, if the number ofbiological elements in a population exceeds the number of pores in thearray, the population can be screened with more than one element in eachpore. The contents of the pores that provide a positive signal can thenbe extracted to provide a subpopulation. The subpopulation can bescreened immediately or, when the subpopulation is cells, it can beexpanded. The screening process can be repeated until each pore of thearray contains only a single element. The screen can also be applied todetect and/or extract the pore that indicates the desired analyte ispresent therein. Following the selection of the pore, other conventionaltechniques may be used to isolate the individual analyte of interest,such as techniques that provide for higher levels of protein production.

After the biological elements have been loaded into the array,additional molecules or particles can be added or removed from the arraywithout disturbing the biological elements. For example, any moleculesor particles useful in the detection of the biological elements can beadded. These additional molecules or particles can be added to the arrayby introducing liquid reagents comprising the molecules or particles tothe top of the array, such as for example by adding drop-wise asdescribed in relation to the addition of the biological elements. Toremove specific molecules from an array comprising biological elements,a solution can be prepared that is free from the selected molecule to beremoved but contains all the rest of the molecules that are present inthe pore array at the desired concentration. The droplet is added to thearray as previously described. After the contents of the pore arrayequilibrate with the droplet of this solution, the concentration of theselected molecule in the array will be reduced. The reduction amountdepends on the volume of the added drop and the total volume containedin the array. To further reduce the concentration of the selectedmolecule, this step may be repeated after removing the first drop fromthe top of the array and then adding a second drop of liquid. Liquid canbe removed from the top of the array by, for example, blotting the arraywith a paper towel or with a pipette.

In certain embodiments, the top of the array is sealed with a membranefollowing the addition of sample to the pores in order to reduceevaporation of the media from the pores. One or more substantially gasand/or liquid impermeable membranes can be used to seal the surfaces ofthe array following the addition of a sample to the pores. For example,typical food-service type plastic wraps such as polyvinylidene fluoride(PVDF) are suitable. In another embodiment, the membrane allows watervapor to equilibrate with the top liquid layer of the liquid in thepore, which can help prevent evaporation. For example, a film placed incontact with the top surface of the micropore array, with water place ontop of the film, would trap the contents of the pores within eachindividual pore, but would allow water or media to flow into the pores.Examples of useful members are nitrocellulose and NAFION® membranes. Asimilar arrangement could be obtained with a porous form of apolytetrafluoroethylene membrane (e.g., GORE-TEX® fabrics) having verysmall holes (e.g., 10-100 nm) that would trap any cells in the pores butallow water, media and other reagents to pass into the pores.

Extraction of Microcavity Content

Based on the optical information received from a detector associatedwith the array of cavities, target cavities with the desired propertiesare identified and their contents extracted for furthercharacterizations and expansion. The disclosed methods are advantageousbecause they maintain the integrity of the biological elements in thecavities. Therefore the methods disclosed herein provide for the displayand independent recovery of a target population of biological elementsfrom a population of up to billions of target biological elements. Thisis particularly advantageous for embodiments where cells are screened.

For example, after pulling the particles to the top or bottom of thepores, the signals from each pore are scanned to locate the bindingevents of interest (See FIG. 4). This identifies the pores of interest.Individual pores containing the desired clones can be extracted using avariety of methods. For all extraction techniques, the extracted cellsor material can be expanded through culture or amplification reactionsand identified for the recovery of the protein, nucleic acid or otherbiological element. As described above, multiple rounds of screening arealso contemplated. Following each screening, one or more cavities ofinterest can be extracted as described herein. The contents of eachcavity can then be screened again until the desired specificity isachieved. In certain embodiments, the desired specificity will be asingle biological element per pore. In these embodiments, extraction mayfollow each round of the screening before the cavities include only asingle element.

In one embodiment, the method includes isolating cells located in themicropores by pressure ejection. For example, a separated micro-porearray is covered with a plastic film. In one embodiment, the methodfurther provides a laser capable of making a hole through the plasticfilm, thereby exposing the spatially addressed micro-pore. Subsequently,exposure to a pressure source (e.g., air pressure) expels the contentsfrom the spatially addressed micro-pore. See WO2012/007537.

Another embodiment is directed to a method of extracting a solutionincluding a biological element from a single microcavity in amicrocavity array. In this embodiment, the microcavity is associatedwith an electromagnetic radiation absorbent material so that thematerial is within the cavity or is coating or covering the microcavity.Extraction occurs by focusing electromagnetic radiation at themicrocavity to generate an expansion of the sample or of the material orboth or evaporation that expels at least part of the sample from themicrocavity. The electromagnetic radiation source may be the same ordifferent than the source that excites a fluorescent label (See FIG. 1,which depicts the same electromagnetic radiation source for bothexcitation of the label and extraction of components of a cavity of thearray). The source may be capable of emitting multiple wavelengths ofelectromagnetic radiation in order to accommodate different absorptionspectra of the materials and the labels.

Subjecting a selected microcavity to focused electromagnetic radiationcan cause an expansion of the electromagnetic radiation absorbentmaterial, which expels sample contents onto a substrate for collectingthe expelled contents. For example, FIG. 9 shows the extraction of aparticular microcavity of interest. Cavities of interest are selectedand then extracted by focusing a 349 nm solid state UV laser at 20-30%intensity power. In one example, the source is a frequency tripled,pulsed solid-state Nd:YAG or Nd:YVO4 laser source emitting about 1microJoule to about 1 milliJoule pulses in about a 50 nanosecond pulse.Both continuous wave lasers with a shutter and pulsed laser sources canbe used. The laser should have sufficient beam quality so that it can befocused to a spot size with a diameter roughly the same or smaller thanthe diameter of the pore. Power levels will depend on the size of thecavity and its contents.

As understood by one of skill in the art, the electromagnetic radiationemission spectra from the electromagnetic radiation source must be suchthat there is at least a partial overlap in the absorption spectra ofthe electromagnetic radiation absorbent material associated with thecavity. The extracted contents can then be located on the capturesurface (See FIG. 9). When the extracted contents include cells, theycan be expanded to obtain the protein, antibody or other element ofinterest.

In some embodiments, the capture surface comprises a hygroscopic layerupon which the contents of the cavity are expelled. The hygroscopiclayer attracts water and prevents the deformation of the optical surfaceallowing clear imaging of the cavity contents. In certain embodiments,the layer is a hygroscopic composition, such as solution comprisingglycerol. The layer can be applied, for example, by spreading, wiping orspraying and should create a uniform dispersion on the surface.Typically, the layer is about 10-100 μm thick, as long as the layer doesnot distort the EM radiation passing through the layer and does nottouch the array above.

Materials within the cavity can be, for example, the particles used inthe binding assays as described above. Accordingly, the particles mayhave a property that allows the particles to respond to a force in orderto accumulate at a detection surface, and also include anelectromagnetic radiation absorbent material, e.g., DYNABEAD® particles.In various embodiments, energy is applied to the particles while theyare accumulated at the detection surface after the signal at thedetection surface is detected (by continued or reapplication of aforce), or the force is removed so that the particles return to thesample solution. Alternatively, the cavities include particles or othermaterials that do not participate in the binding reactions but arepresent to provide extraction of the contents as described herein. Theseparticles may be functionalized so that they bind to the walls of themicro-cavities independent of the binding reaction of the assay. Similarmaterials can be used to coat or cover the micropores, and inparticular, high expansion materials, such as EXPANCEL® coatings(AkzoNobel, Sweden). In another embodiment the EXPANCEL® material can besupplied in the form of an adhesive layer that is bonded to one side ofthe array so that each pore is bonded to an expansion layer.

Focusing electromagnetic radiation at a microcavity can cause theelectromagnetic radiation absorbing material to expand, which causes atleast part of the liquid volume of the cavity to be expelled. Heatingthe material may also cause a rapid expansion of the contents of thecavity. In some embodiments, a portion of the of the contents (up to50%) are expanded up to 1600 times by heating, which causes a portion ofthe remainder of the contents to be expelled from the cavity.

Without rapid expansion of the material or cavity contents, heating cancause evaporation of the contents, which can be collected by condensingthe contents on a substrate. For example, the substrate can be ahydrophobic micropillar placed at or near the opening of the cavity.Expulsion of the contents may also occur as the sample evaporates andcondenses on the walls of a capillary outside the meniscus, which causesthe meniscus to break and release the contents of the capillary.

Micro-cavities, such as a micropore, can be open at both ends, with thecontents being held in place by hydrostatic force. During the extractionprocess, one of the ends of the cavities can be covered to preventexpulsion of the contents from the wrong end of the cavity. The cavitiescan be covered in the same way as, for example, the plastic filmdescribed above. Also, the expansion material may be bonded as a layerto one side of the array.

Apparatus

Detection of analytes in accordance with the invention requires, in someembodiments, the use of an apparatus capable of applying electromagneticradiation to the sample, and in particular, to an array of vessels, suchas a microarray. The apparatus must also be capable of detectingelectromagnetic radiation emitted from the sample, and in particularfrom the detection surface of the sample vessel.

Any type of electromagnetic radiation source may be used according tothe disclosure without departing from the scope of the invention. In oneembodiment, the electromagnetic radiation source of the apparatus isbroad spectrum light or a monochromatic light source having a wavelengththat matches the wavelength of at least one label in a sample. In afurther embodiment, the electromagnetic radiation source is a laser,such as a continuous wave laser. In yet a further embodiment, theelectromagnetic source is a solid state UV laser. A non-limiting list ofother suitable electromagnetic radiation sources include: argon lasers,krypton, helium-neon, helium-cadmium types, and diode lasers. In someembodiments, the electromagnetic source is one or more continuous wavelasers, arc lamps, or LEDs.

In some embodiments, the apparatus comprises multiple electromagneticsources. In other embodiments, the multiple electromagnetic (EM)radiation sources emit electromagnetic radiation at the samewavelengths. In other embodiments, the multiple electromagnetic sourcesemit different wavelengths in order to accommodate the differentabsorption spectra of the various labels that may be present in thesample.

In some embodiments, the electromagnetic radiation source is locatedsuch that an array of micropores can be contacted with electromagneticradiation. In some embodiments, the electromagnetic radiation source islocated such that the detection surface of a vessel can be subjected toelectromagnetic radiation.

The apparatus also includes a detector that receives electromagnetic(EM) radiation from the label(s) in the sample, array. The detectors canidentify at least one vessel (e.g., a micropore) emittingelectromagnetic radiation from one or more labels.

In one embodiment, light (e.g., light in the ultra-violet, visible orinfrared range) emitted by a fluorescent label after exposure toelectromagnetic radiation is detected. The detector or detectors arecapable of capturing the amplitude and duration of photon bursts from afluorescent moiety, and further converting the amplitude and duration ofthe photon burst to electrical signals.

Once a particle is labeled to render it detectable, or if the particlepossesses an intrinsic characteristic rendering it detectable, anysuitable detection mechanism known in the art may be used withoutdeparting from the scope of the present invention, for example a CCDcamera, a video input module camera, a Streak camera, a bolometer, aphotodiode, a photodiode array, avalanche photodiodes, andphotomultipliers producing sequential signals, and combinations thereof.Different characteristics of the electromagnetic radiation may bedetected including: emission wavelength, emission intensity, burst size,burst duration, fluorescence polarization, and any combination thereof.

The detection process can also be automated, wherein the apparatuscomprises an automated detector, such as a laser scanning microscope.

In some embodiments, the apparatus as disclosed can comprise at leastone detector; in other embodiments, the apparatus can comprise at leasttwo detectors, and each detector can be chosen and configured to detectlight energy at the specific wavelength range emitted by a label. Forexample, two separate detectors can be used to detect particles thathave been tagged with different labels, which upon excitation with anelectromagnetic source, will emit photons with energy in differentspectra.

Kits

In another aspect, the present invention is directed to kits fordetecting an analyte. In exemplary embodiment, the kits include amicropore array of plurality of longitudinally fused capillaries have adiameter of about 1 micrometer to about 500 micrometers. In certainembodiments, the capillaries minimize light or EM radiation transmissionbetween capillaries. The kits may also include particles that are ableto move within a sample liquid when subjected to motive force, such asmagnetic particles. These particles may include a binding partner forthe analyte or other sample component. In certain embodiments, the kitsinclude magnetic particles that have been functionalized with a bindingpartner for the analyte. In addition, the kit can optionally include oneor more labels capable of emitting electromagnetic radiation, whereinthe labels may be conjugated to a binding partner for the analyte orother sample component. The kits may also include second, third, orfourth, etc. set of particles that are functionalized to bind second,third, or fourth, etc. analytes in the sample, or to provide for thenormalization of the sample components between micropores of the array.Stabilizers (e.g., antioxidants) to prevent degradation of the reagentsby light or other adverse conditions may also be part of the kits. Inother embodiments, the kits contain antibodies specific for a proteinexpressed from a gene of interest, in addition to detection reagents andbuffers. In other embodiments, the kits contain reagents specific forthe detection of mRNA or cDNA (e.g., oligonucleotide probes or primers).

The kits may optionally include instructional materials containingdirections (i.e., protocols) providing for the use of the micro-porearray in the detection of various biological compounds that are secretedfrom a biological cell. While the instructional materials typicallyinclude written or printed materials they are not limited to such. Anymedium capable of storing such instructions and communicating them to anend user is contemplated by this invention. Such media include, but arenot limited to electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD ROM), and the like. Suchmedia may include addresses to internet sites that provide suchinstructional materials.

In other embodiments, the present invention provides kits for thedetection, identification, and/or characterization of proteins and/ornucleic acids. In some embodiments, the kits contain antibodies specificfor an analyte, such as a protein of interest, in addition to detectionreagents and buffers. In other embodiments, the kits contain reagentsspecific for the detection of mRNA or cDNA (e.g., oligonucleotide probesor primers). In various embodiments, the kits contain all of thecomponents necessary to perform a detection assay, including allcontrols, directions for performing assays, and any necessary softwarefor analysis and presentation of results

Biological Recognition Assays

Although it is not necessary to understand the mechanism of aninvention, it is believed that the arrays described above comprisemicro-pores having sufficient volume to incubate the cells for between0-240 hours, such that compounds of interest are secreted from the cellsand bind to the binding partner. Consequently, a plurality of biologicalrecognition assays may be performed either within, or between, each ofthe micro-pores. For example, one such recognition assay may compriseantigen-antibody binding.

In one embodiment, the present invention contemplates a method forantigen-antibody binding comprising incubating a plurality of cells at37° C. for 1-24 hours such that each cell produces antibodies andsecretes the antibodies into the micro-pore. In one embodiment, theantibody is a recombinant antibody. In one embodiment, the antibody is amonoclonal antibody. In one embodiment, at least one of the cellsproduces more than one antibody. Although it is not necessary tounderstand the mechanism of an invention, it is believed that because ofthe micropore architecture, this incubation has reduced evaporationlosses due to the very narrow inlets (thus small exposed surface area)and the extreme length of the pores. In one embodiment, the antibodysecretion is stimulated by an induction agent. In one embodiment, theinduction agent comprises IPTG. See, FIG. 3.

Therapeutic Drug Discovery

In one embodiment, the present invention contemplates a methodcomprising identifying new therapeutic drugs. For example, particles maybe coated with a drug binding partner known to be involved in a diseasecondition (i.e., for example, a biological receptor and/or enzyme). Aplurality of cells secreting various compounds suspected of havingaffinity for the binding partner is then screened using the veryhigh-density micro-pore array. The micro-pores containing the particlesattached to binding partner-compound complexes having the highestaffinity are selected for future development.

Diagnostic Antibody Discovery

In one embodiment, the present invention contemplates a methodcomprising identifying diagnostic antibodies. For example, particles maybe coated with a binding partner known to be involved in a diseasecondition (i.e., for example, an antigen and/or epitope). A plurality ofcells secreting various antibodies suspected of having affinity for thebinding partner is then screened using the very high-density micro-porearray. The micro-pores containing particles the binding partner-antibodycomplexes having the highest affinity are selected for futuredevelopment.

Protein-Protein Interaction Studies

In one embodiment, the present invention contemplates a methodcomprising identifying protein-protein interactions. For example,particles may be coated with a binding partner known to be involved in adisease condition (i.e., for example, a protein and/or peptide). Aplurality of cells secreting various proteins and/or peptides suspectedof having affinity for the binding partner is then screened using thevery high-density micro-pore array. The micro-pores containing particleswith immobilized binding partner-protein or peptide complexes having thehighest affinity are selected for future development.

Protein-Nucleic Acid Interaction Studies

In one embodiment, the present invention contemplates a methodcomprising identifying protein-nucleic acid interactions. For example,particles may be coated with a binding partner known to be involved in adisease condition (i.e., for example, a deoxyribonucleic acid and/or aribonucleic acid and/or a SOMAmer and/or a Apatamer). A plurality ofcells secreting various proteins and/or peptides suspected of havingaffinity for the binding partner is then screened using the veryhigh-density micro-pore array. The micro-pores containing particles withimmobilized binding partner-nucleic acid complexes having the highestaffinity are selected for future development.

Protein-Carbohydrate Interaction Studies

In one embodiment, the present invention contemplates a methodcomprising identifying protein-carbohydrate interactions. For example,particles may be coated with a binding partner known to be involved in adisease condition (i.e., for example, an oligosaccharide, andliposaccharide, or a proteosaccharide). A plurality of cells secretingvarious lectins, proteins and/or peptides suspected of having affinityfor the binding partner is then screened using the very high-densitymicro-pore array. The micro-pores containing particles with immobilizedbinding partner-carbohydrate complexes having the highest affinity areselected for future development.

Detection of Protein

Disclosed herein is the use of a highly sensitive system to identify aprotein analyte from a heterologous population of biological elements.The disclosed methods and system provide a novel and important tool forprotein engineering. In particular, the uses disclosed herein shouldlead to a significant reduction in time of new antibody discovery, from14 days to 1-2 days. In other embodiments, gene expression may bedetected by measuring the expression of a protein or polypeptide.

In one embodiment, the disclosed method is used to detect a proteinanalyte. The detection of a particular analyte protein can be performeddirectly in the pores of the array. Images are taken after the particlesare accumulated at the detection surface of the pore, such that thebackground signal is inhibited.

The biochemical sensing can be done using standard detection techniquesincluding a sandwich immunoassay or similar binding or hybridizingreactions. In conjunction with the magnetic particle the media is loadedwith secondary antibodies that are optically labelled (e.g., attached toflourochromes).

The secondary antibodies bind to the particle if the target proteinproduced by the elements binds to the antigen that is attached to theparticle surface (See FIG. 3A). To maximize the binding capability ofthe target protein to the antibodies, the magnetic particles are kept insuspension by periodically (for example, approximately every 20 min.)exposing them to a magnetic field such that the particles are circulatedthroughout the liquid contained in the sample volume.

Detection of Nucleic Acids

In one embodiment, the analyte is a nucleic acid. For example, DNA ormRNA expression may be measured by any suitable method, For example, RNAexpression is detected by enzymatic cleavage of specific structures(INVADER® assay, Third Wave Technologies; See, e.g., U.S. Pat. Nos.5,846,717, 6,090,543; 6,001,567; 5,985,557; and 5,994,069; each of whichis herein incorporated by reference). The INVADER® assay detectsspecific nucleic acid (e.g., RNA) sequences by using structure-specificenzymes to cleave a complex formed by the hybridization of overlappingoligonucleotide probes.

In still further embodiments, RNA (or corresponding cDNA) is detected byhybridization to an oligonucleotide probe. A variety of hybridizationassays using a variety of technologies for hybridization and detectionare available. For example, in some embodiments, TAQMAN® assay (PEBiosystems, Foster City, Calif.; See, e.g., U.S. Pat. Nos. 5,962,233 and5,538,848, each of which is herein incorporated by reference) isutilized. The assay is performed during a PCR reaction. The TaqMan assayexploits the 5′-3′ exonuclease activity of the AMPLITAQ® GOLD DNApolymerase. A probe consisting of an oligonucleotide with a 5′-reporterdye (e.g., a fluorescent dye) and a 3′-quencher dye is included in thePCR reaction. During PCR, if the probe is bound to its target, the 5′-3′nucleolytic activity of the AMPLITAQ® GOLD polymerase cleaves the probebetween the reporter and the quencher dye. The separation of thereporter dye from the quencher dye results in an increase offluorescence. The signal accumulates with each cycle of PCR and can bemonitored with a fluorimeter.

In yet other embodiments, reverse-transcriptase PCR (RT-PCR) is used todetect the expression of RNA. In RT-PCR, RNA is enzymatically convertedto complementary DNA or “cDNA” using a reverse transcriptase enzyme. ThecDNA is then used as a template for a PCR reaction. PCR products can bedetected by any suitable method, including but not limited to, gelelectrophoresis and staining with a DNA specific stain or hybridizationto a labeled probe. In some embodiments, the quantitative reversetranscriptase PCR with standardized mixtures of competitive templatesmethod described in U.S. Pat. Nos. 5,639,606, 5,643,765, and 5,876,978(each of which is herein incorporated by reference) is utilized.

Detection of Cells

In another embodiment, the analyte is a biological cell. In oneembodiment, the analyte is at least one biological cell.

The detection disclosed herein may be used to isolate any types ofbiological cells, including, but not limited to, cell lines that expressor produce proteins, carbohydrates, enzymes, peptides, hormones,receptors; other cell lines that produce antibodies; geneticallyengineered cells; and/or activated cells. Moreover, the presentinvention may be used to screen for a variety of biological activitiesincluding, but not limited to, the expression of surface receptorproteins, enzyme production, and peptide production. Furthermore, thepresent invention may be used to screen a variety of test agents todetermine the effect of the test agents on the desired biologicalactivity. Other types of cells desired to be isolated and screened,other types of biological activity desired to be detected, and specifictest agents to be screened will be readily appreciated by one of skillin the art.

In a particular embodiment, the biological cell is a transformedbiological cell. Transformation of cells can occur by any well-knownmethods, using any well-known vectors, such as for example a plasmid orvirus.

In one embodiment, the biological cell is a microbial, fungal,mammalian, insect or animal cell. In one embodiment, the microbial cellis a bacterial cell. In one embodiment, the bacterial cell is an E. colicell.

In one embodiment, the animal cell includes a rare biochemical compound.In one embodiment, the rare biochemical compound is selected from thegroup comprising a protein, a peptide, a hormone, a nucleic acid, acarbohydrate.

In another embodiment, the biological cell produces and/or expresses afluorescent protein. In yet another embodiment, the biological cellproduces fluorescent protein (eg. GFP).

EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1: Magnetic Bead Signal Blocking

This example demonstrates the ability of the magnetic beads to inhibitthe high background signal from labels in solution that are not bound tothe particles.

A clean and dry 20 μm pore diameter array plate (J5022-19, HamamatsuPhotonics K. K.) was loaded with 10 μM Alexa 488 flourochrome (SigmaAldrich) as a label and 2.8 μm magnetic particles such that each porereceives both labels and magnetic particles. The fluorochrome labelalone does not bind or associate with the magnetic bead particles.

The pores of the array are not exposed to any motive force prior todetection, so both the magnetic particles and the labels both remain insolution. Following incubation, this array comprising the magneticparticles and labels is subjected to EM radiation and the fluorescentemission from the label is imaged using a microscope. As expected, asshown in FIG. 5A, all of the pores show significant signal, for theunbound labels in solution have full exposure to the EM radiation. Thefluorescent images were captured using a CCD camera. When the pixelcount and intensity was quantified, the signal shows a pixel count atthe appropriate intensity of about 4×10⁵. Therefore, in the absence ofparticle accumulation, the unbound labels in solution will emit signalin nearly all of the pores regardless of their content.

Next, in the same array, the magnetic particles were accumulated to thedetection surfaces of each reaction vessel of the array (e.g., meniscusin this example) by placing a 10 mm cubed neodymium magnet (B666, K&JMagnetics Inc.) at the bottom of the array (within 3 mm) for 60 seconds.After the particles have accumulated, another fluorescent image iscaptured of the same location. As shown in FIG. 5B, the image shows astriking decrease in pores with signal. Indeed, nearly all of the poreshave lost or reduced their signal. When the signal is quantified, thesignal at the appropriate intensity in the accumulated array drops tozero. The signal from the unbound labels is inhibited by the magneticbeads and only the basal autofluorescence from the array remains.Surprisingly, the accumulation of particles at the detection surface ofthe sample represents a novel strategy for eliminating background signalfrom unbound labels each pore.

Example 2: Assay Preparation

This example provides a representative assay system for detecting thepresence or absence of an analyte in an array. The final screeningdesign will, of course, depend on many factors specific to theexperiment and its objectives.

The E. coli strain Imp4213, which contains a plasmid expressing GFP, wasused in this assay. Approximately 1.0 to 10 million GFP-expressingbacterial cells were added into 5 mL of Luria broth (LB) media with 25μg/ml kanamycin (KAN) and expanded on a shaker (200 RPM) at 37° C. forapproximately 3 hours until the bacteria entered the exponential growthphase, and then were prepared for plating.

The particles used in this example are magnetic beads. Specifically, forthese examples, the particles used were 2.8 μm diameter magnetic beads(Dynabeads) coupled to a either streptavidin (Part No. 112-05D,Invitrogen, Carlsbad, Calif.) or oligo(dt)₂₅ ligands (Part No. 610-05,Invitrogen, Carlsbad, Calif.). The magnetic beads were incubated with abiotinylated rabbit anti-GFP antibody (A10259, Invitrogen, Carlsbad,Calif.) for about 30 minutes, which bound the anti-GFP antibody to themagnetic beads via the biotin-streptavidin interaction. Therefore theanti-GFP magnetic beads were generated.

In this example, before the cells are added to the array, the cells aresupplemented with the magnetic beads and labels. The magnetic beads(either anti-GFP beads to detect analyte or oligo(dt)₂₅ beads as anegative control) were added to a concentration of 30 mg/ml. The labelis generated by combining 15 μg/ml biotinylated rabbit anti-GFP (A10259,Invitrogen, Carlsbad, Calif.) with fluorophore Atto 590 conjugated tostreptavidin (40709-1MG-F, Sigma Aldrich), which forms afluorescently-labeled anti-GFP antibody. For this assay design, anyfluorophore label could be used, as long as it does not emit EMradiation around the same wavelength as GFP.

The cells were added to a clean and dry 20 μm pore diameter array plate(J5022-19, Hamamatsu Photonics K. K.) by placing a drop onto the arrayand spreading the analysis mixture on to the array plate surface toapproximately 1 μl/mm² (FIG. 3). The mixture is spontaneouslydistributed into the pores through the hydrophilic pore surface.

Once the sample is distributed, the array plate is placed on a holdersuch that the pore array plate is 80 μl above the capture surface(Sylgard® 184 Silicone Elastomer Kit, Dow Corning). The holder iscomposed of a glass slide (12-550-14, Fisher Scientific) with a 250 μmthick capture surface, two 80 μm adhesive tape spacers on either side ofthe capture surface. Once the pore array is placed on the holder, theupper side of the array is sealed with plastic wrap (22-305654, FisherScientific). The full setup is placed into a 100 mm petri dish with two10 mm deionized water-wet tissue paper spheres for humidity saturation.The dish is sealed with parafilm and allowed to incubate at 37° C. for19 hours.

To select for pores with the analyte, the magnetic beads are accumulatedat the bottom surface of the pore array by placing a 10 mm cubedneodymium magnet (B666, K&J Magnetics Inc.) at the bottom of the array(within 3 mm) for 60 seconds. The array on the holder is then loadedinto an Arcturus Laser Capture Microdissection System (Model: Veritas,Applied Biosystems, USA) to facilitate detection.

To detect fluorescence of pores containing analyte, the immunoassayresults are scanned by reading the bottom surface fluorescence of thepore array plate with a microscope comprising a 20× objective and a 594nm excitation 630 nm emission fluorescent cube. Image capture wasperformed using a CCD camera.

Example 3: Detection of Analyte in Array

This example illustrates that a protein binding and detection assay canbe performed directly in the pores using the methods disclosed herein.In this embodiment, the assay system is set up according to the stepsdescribed in Example 2.

The first array contains pores comprising the anti-GFP magneticparticles and the anti-GFP labels generated as described in Example 2.After incubation, the array is exposed to a magnetic force, asdescribed, moving the particles to the detection surface of the sample,which in this example is the meniscus of each vessel of the array. Animage of this array is captured. The anti-GFP magnetic particles boundto the target protein (GFP), which is also bound by the anti-GFP label.The magnetic forces move the labeled particles to the detection surface,therefore concentrating the signal-emitting labels at the detectionsurface, and inhibiting the unbound labels from transmitting EMradiation to the detector or microscope. A schematic diagram of thisinteraction within the pore is shown in FIG. 1B.

The second array contains particles that are bound to ligand oligo-dTrather than streptavidin. Without streptavidin, the biotin conjugated tothe anti-GFP antibody does not interact or bind with the magneticparticles. Therefore both the magnetic particles, anti-GFP antibodies,and anti-GFP labels remain in solution. After incubation, the array isexposed to magnetic force, moving the particles to the detectionsurface. The array is imaged following EM radiation exposure. Since theoligo-dT magnetic particles are not bound to any label, they accumulateto the detection surface and inhibit the EM radiation from reaching thelabel in solution. The particles also can be used to inhibit thedetector from detecting the EM radiation emitted from the labels unboundto the magnetic particles. If the amount of fluorescence from each arrayis quantified, the emission from the specific anti-GFP magnetic beads issubstantially higher than the negative control oligo(dt) beads.

Therefore, this experiment shows that the magnetic bead inhibitingmethods as disclosed can select analyte-containing pores by specificinhibiting of pore signal.

Example 3: Single Cell Specific Detection

The following example demonstrates the specificity of the disclosedmethods for detecting analytes at the single cell level.

In this embodiment, the assay system is set up according to the stepsdescribed in Example 2, except that before the cells are plated, thebacterial cells were diluted to 300 cells per microliter, which wascalculated to result in approximately 0.1 cell per pore on the arrayplate with pores having a 20 micrometer diameter. When added to thearray, this dilution will yield up to a single bacterial cell in mostpores.

The anti-GFP streptavidin beads and anti-GFP label were added to thefirst array as described. The corresponding signal seen when EMradiation is exposed to the array following magnetic bead accumulationis shown in FIG. 7A. Many cells are signal-producing, which representsfluorescent signal from streptavidin magnetic beads that are bound tobiotinylated anti-GFP antibody via streptavidin-biotin linkage. Afterapplying magnetic force, only cells that express the analyte targetprotein (GFP) and therefore bind the beads to the label provide positivesignal. However, also observed with single cell dilutions are pores withreduced or absent signal. These pores most likely lack any GFP-producingcells and/or contain bacterial cells that no longer express GFP.Therefore no label is bound to magnetic particle, and upon applicationof magnetic force, the magnetic particles inhibit the detection surface.

Accordingly, the other array comprising oligo(dt)₂₅ beads depicts verylittle fluorescent signal from pores, as shown in FIG. 7B. In thisarray, there is no anti-GFP antibody bound to the beads, and thereforeno label bound to the beads. After applying magnetic force, none of thecells that express the analyte target protein (GFP) will provide signal,since the magnetic beads have accumulated to the detection surfacewithout a label. In this setup, the EM radiation from the EM sourcecannot reach the unbound label in solution for these pores. Therefore,no signal is observed in this array. The magnetic beads could also beaccumulated between the sample and the detector to inhibit emission fromthe label to the detector.

Example 4: Detection does not Require Lysis of Cells

This example shows that performing a sandwich immunoassay with beads inthe array does not require cell lysis for analyte detection.

In these embodiments, the arrays were prepared as described in theprevious examples with the two positive control arrays containingmagnetic beads linked to anti-GFP antibody. The cells of one positivecontrol array were lysed using sonication for 1.5 minutes at 5 wattsprior to detection using a Misonix 4000 with Microplate horn, while theother positive control array was not lysed.

The negative control array contains Oligo-dT beads, which earlierexamples have shown inhibit the signal from all pores in this arraysystem. The negative control array was lysed using the same sonicationconditions as the positive control array. The results indicate (see FIG.8A-C) that both of the positive control arrays have similar signallevels regardless of cell lysis (sonication) step, based on thequantification of pixel count. Further, the lysis step did not removethe selectivity of the particle inhibiting, for the negative controlbeads still remain far below the positive control levels. FIGS. 8A and8B. This suggests that for certain expression mechanisms cell lysis isnot required to detect analytes using the methods described herein.

Example 5: Extraction and Recovery of Cells

The following example demonstrates the display and independent recoveryof a single target among billions of target cells.

In this example, the cells, beads, labels, and arrays are prepared asdescribed in Example 2. After identification of one or more porescontaining analyte, extraction of the solution in the single pore isdesired.

As described and depicted in FIG. 9, the array is placed on a holdersuch that the pore array is about 80 micrometers above the capturesurface. The holder is composed of a glass slide (12-550-14, FisherScientific) with a 250 μm thick capture surface, two 80 μm adhesive tapespacers on either side of the capture surface. Once the pore array isplaced on the holder the upper side of the array is sealed with plasticwrap (22-305654, Fisher Scientific).

For recovery, a pore of interest is selected, such as a high intensitypore. The pore is then extracted by focusing a 349 μm solid state UVlaser at 20 to 30 percent intensity power and emmitting a 20 microJoulepulse. The cells extracted from the targeted pore can then be located onthe capture surface, as shown in FIG. 9.

After extraction, the holder and the array are then removed from theArcturus Laser Capture Microdissection System and the capture surfacecontaining the extracted cells is placed into 5 mL LB plus 25 μg/ml KANmedia and incubated on a shaker at 37° C. for 19 hours to expand theextracted cells. These cells, which express the protein of interest, arethen ready for any type of scale-up protein production and/or furtherprocessing.

Example 6: Modifying Reagent Concentration in Pores of the Array

The following example demonstrates that reagents and other molecules,such as magnetic beads, can be added to the array after it is loadedwithout disturbing the cells within the array.

In this embodiment, Freestyle 293 Media containing Human EmbryonicKidney 293 (HEK 293) cells was added at a concentration of 2,500cells/μL into a micro-pore array with a pore diameter of 100 μm. Thisloaded about 20 cells per pore, with a cell viability of about 50%. Thecells were first imaged with a fluorescent microscope at 10× using aTexas Red filter cube to measure any background fluorescence in thatchannel (FIG. 10A). Next, a 50 μL drop of Freestyle Media with 2 μg/mLpropidium iodide (PI) was placed on top of the micropore array. At 660Daltons, PI is a relatively small molecule and diffuses about 400 μm in60 seconds in an aqueous solution at room temperature. Because themicropores are approximately 1 mm long, the loaded cells at the bottomof the micropores should be exposed to PI molecules after about 5minutes. This was confirmed by detecting the intercalation of the PImolecules with the DNA in the nucleuses of the non-viable HEK 293 cellsas shown in FIG. 10B.

ADDITIONAL EMBODIMENTS

The following additional embodiments are non-limiting and presented hereto further exemplify various aspects of the disclosure.

An apparatus for detecting a target biological element comprising:

-   -   an array of micropores having an internal diameter of about 1        micrometer to about 500 micrometers,    -   a magnetic source that applies a magnetic field to the array,    -   an electromagnetic radiation source for applying electromagnetic        radiation to the array, and    -   a detector that receives electromagnetic radiation from the        array and identifies the location of at least one micropore        emitting the radiation.

The apparatus further comprises magnetic particles comprising a bindingpartner in each micropore of the array.

The apparatus further comprises an energy source that focuses energy onthe micropores that emit electromagnetic radiation.

The micropores of the apparatus are not translucent.

The micropores are coated with a material that prevents lighttransmission between the pores.

A kit for detecting an analyte, comprising: a micropore array comprisinga plurality of longitudinally fused fibers having a diameter of about 1micrometer to about 500 micrometers, and magnetic particles comprising abinding partner for the analyte.

The micropores are not translucent.

The micropores are coated with a material that prevents lighttransmission between the pores.

Although various specific embodiments of the present invention have beendescribed herein, it is to be understood that the invention is notlimited to those precise embodiments and that various changes ormodifications can be affected therein by one skilled in the art withoutdeparting from the scope and spirit of the invention.

It is understood that the invention is not limited to the particularmethodology, protocols, and reagents, etc., described herein, as thesemay vary as the skilled artisan will recognize. It is also to beunderstood that the terminology used herein is used for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the invention.

It should be noted that the features illustrated in the drawings are notnecessarily drawn to scale, and features of one embodiment may beemployed with other embodiments as the skilled artisan would recognize,even if not explicitly stated herein.

Any numerical values recited herein include all values from the lowervalue to the upper value in increments of one unit provided that thereis a separation of at least two units between any lower value and anyhigher value. As an example, if it is stated that the concentration of acomponent or value of a process variable such as, for example, size,angle size, pressure, time and the like, is, for example, from 1 to 90,specifically from 20 to 80, more specifically from 30 to 70, it isintended that values such as 15 to 85, 22 to 68, 43 to 51, 30 to 32,etc. are expressly enumerated in this specification. For values whichare less than one, one unit is considered to be 0.0001, 0.001, 0.01 or0.1 as appropriate. These are only examples of what is specificallyintended and all possible combinations of numerical values between thelowest value and the highest value enumerated are to be considered to beexpressly stated in this application in a similar manner.

The disclosures of all references and publications cited herein areexpressly incorporated by reference in their entireties to the sameextent as if each were incorporated by reference individually.

The invention claimed is:
 1. A method for detecting a target biologicalelement from a sample comprising a heterogeneous population ofbiological elements, comprising: distributing the heterogeneouspopulation of biological elements into an array of receptacles, whereineach of the receptacles comprises a microcavity; adding, to the array,particles and a label that emits electromagnetic radiation uponactivation, wherein the label is bound to the particles or the labelbecomes bound to the particles as a result of the presence or absence ofthe target biological element in the sample; applying a force to thearray to accumulate the particles at a surface of a portion of thesample in each of the receptacles, wherein the surface of the portion ofthe sample is a meniscus, and identifying the presence or amount ofelectromagnetic radiation emitted from the receptacles, therebyidentifying receptacles containing the target biological element.
 2. Themethod of claim 1, wherein the label is released from the particles as aresult of the presence or absence of the target biological element inthe sample.
 3. The method of claim 1, wherein the biological element isan organism, cell, protein, nucleic acid, lipid, saccharide, metabolite,or small molecule.
 4. The method claim 3, wherein the cell is atransformed cell that produces a recombinant protein.
 5. The method ofclaim 4, wherein the recombinant protein is an enzyme and the label is asubstrate for the enzyme.
 6. The method of claim 3, wherein the cell isa transformed cell that produces a recombinant antibody.
 7. The methodof claim 1, wherein the sample is added to the array at a concentrationthat is intended to introduce no more than a single biological elementin each receptacle.
 8. The method of claim 1, wherein the array ofreceptacles comprises a microcavity array comprising a plurality oflongitudinally fused capillaries having a diameter of about 1 to about500 micrometers.
 9. The method of claim 1, wherein the array comprisesbetween about 300 and 64,000,000 capillaries per square centimeter ofthe array.
 10. The method of claim 1, wherein the sidewalls of themicrocavity of each of the receptacles are not translucent.
 11. Themethod of claim 1, wherein reagents are added to the array byintroducing fluids comprising the reagents to the top of the array. 12.A method for detecting a target biological element from a samplecomprising a heterogeneous population of biological elements,comprising: distributing the heterogeneous population of biologicalelements into an array of receptacles, wherein the array of receptaclescomprises a microcavity array comprising a plurality of longitudinallyfused capillaries having a diameter of about 1 to about 500 micrometers;adding, to the array, particles and a label that emits electromagneticradiation upon activation, wherein the label is bound to the particlesor the label becomes bound to the particles as a result of the presenceor absence of the target biological element in the sample; applying aforce to the array to accumulate the particles at a surface of theportion of the sample in each of the receptacles, and identifying thepresence or amount of electromagnetic radiation emitted from thereceptacles, thereby identifying receptacles containing the targetbiological element.
 13. The method of claim 12, wherein the label isreleased from the particles as a result of the presence or absence ofthe target biological element in the sample.
 14. The method of claim 12,wherein the biological element is an organism, cell, protein, nucleicacid, lipid, saccharide, metabolite, or small molecule.
 15. The methodof claim 12, wherein the sample is added to the array at a concentrationthat is intended to introduce no more than a single biological elementin each receptacle.
 16. The method of claim 12, wherein the sidewalls ofmicrocavities of the microcavity array are not translucent.
 17. Themethod of claim 12, wherein reagents are added to the array byintroducing fluids comprising the reagents to the top of the array. 18.A method for detecting a target biological element from a samplecomprising a heterogeneous population of biological elements,comprising: distributing the heterogeneous population of biologicalelements into an array of receptacles, wherein the array comprisesbetween about 300 and 64,000,000 capillaries per square centimeter ofthe array; adding, to the array, particles and a label that emitselectromagnetic radiation upon activation, wherein the label is bound tothe particles or the label becomes bound to the particles as a result ofthe presence or absence of the target biological element in the sample;applying a force to the array to accumulate the particles at a surfaceof the portion of the sample in each of the receptacles, and identifyingthe presence or amount of electromagnetic radiation emitted from thereceptacles, thereby identifying receptacles containing the targetbiological element.
 19. The method of claim 18, wherein the label isreleased from the particles as a result of the presence or absence ofthe target biological element in the sample.
 20. The method of claim 18,wherein the biological element is an organism, cell, protein, nucleicacid, lipid, saccharide, metabolite, or small molecule.
 21. The methodof claim 18, wherein the sample is added to the array at a concentrationthat is intended to introduce no more than a single biological elementin each receptacle.
 22. The method of claim 18, wherein the array ofreceptacles comprises an array of microcavities and the sidewalls of themicrocavities are not translucent.